Macromolecules 1993,26, 5809-5811
Composition Range of Crystal Phase Transition of Isodimorphism in Poly(3-hydroxybutyrate-co-thydroxyvalerate) Hiroshi Mitomo' Faculty of Engineering, Gunma University, Kiryu, Gunmn 376, Japan
Norio Morishita Japan Atomic Energy Research Institute, Takasaki Radiation Chemistry Research Establishment, Watanuki, Takasaki-Shi, Gunma 370-12, Japan
Yoshiharu Doi Polymer Chemistry Laboratory, The Institute of Physical and Chemical Research (RZKEN), 2-1, Hirosawa, Wako-Shi, Saitama 351 -01, Japan Received April 27, 1993 Revised Manuscript Received July 26, 1993
Introduction Poly(3-hydroxybutyrate) (P(3HB)) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate)(P(3HBco-3HV))can be isolated with a very high purity from the cells of some bacteria by solvent extraction.lI2 It has been reported that a structural characteristicof P(3HB-co-3HV) is isodimorphism, i.e., P(3HB-co-3HV)crystallizing either in a P(3HB) or P(3HV) crystal lattice for the HV contents lower or higher than ca. 40 mol %, respectively.3~~ Recently,it has been reported that the coexistenceof both crystal phases and pseudoeutectic behavior occur at a narrow composition range, i.e., at a composition of around ) ~ 7% for 32 mol % for synthetic ~ ( ~ H B - C O - ~orH41Vmol bacterialP(~HB-co-~HV).**~ The cocrystallizationof these copolymers has been interpreted theoretically with thermodynamic treatment.gl0 In the present study, the composition range of crystal phase transition, where thepseudoeutectic or bothP(3HB) and P(3HV) crystal phases coexist, was investigated for the samples fractionated with a solution of an acetonewater system by wide-angle X-ray scattering and differential scanning calorimetry. Experimental Section Copolymers P(3HB-co-3HV)containing 34.2,45.7,64.2, and 67.8 mol % HV were used, which were isolated from Alcaligenes eutrophus (ATCC 17699). The microorganism was first grown at 30 "C in nutrient-rich medium (100 mL) containing 1 g of yeast extract, 1g of polypeptone, 0.5 g of meat extract, and 0.5 g of (N&)&O,. The cells were harvested after 24 h and washed with water. At this stage, accumulation of polyesters in the cells was not observed. To promote polyester synthesis, about 0.4-g quantities of the washed cells were transferred into a nitrogenfree medium" containing 2 g of different ratios of two carbon sources, i.e., butyric and valeric acids. T h e cella were cultivated in these media (100 mL, pH. = 7.0) for 48 h at 30 "C, harvested by centrifugation, washed with acetone, and f i y dried under vacuum at room temperature. Polyesters were extracted from the dried cells with hot chloroform in a Soxhlet apparatus and purified by reprecipitation with hexane? The procedure of fractionation of these samples was as follows: the samples were dissolved with hot acetone and then kept at 8 OC for 20 h followed by fitration or centrifugation to isolate the precipitate. Next, the solution was diluted with water to a concentration of 95 % acetone and kept at the same condition 0024-9297/93/2226-5809$04.00/0
5809
followed by isolation. The same procedure was repeated to the concentration, lowering by every 6% step. The melting behavior of a 3-mg sample was studied by using a Perkin-Elmer Model DSC-7 differential scanning calorimeter (DSC)at a heatingrate of 10"Clmin under anitrogen atmosphere. The melting peak temperature, after being corrected for the thermal lag (or heating rate dependence) and calibrated with high-purity standards, was defied as the melting point T , with accuracy within fO.l "C. Thewide-angleX-ray scattering (WAXS) pattern was recorded photographically using a cylindrical vacuum camera (50 mm 4) by the method of rotating the needle-shaped sample. Nickelfiltered Cu Ka X-ray beams with a pinhole collimator from a Rigaku-DenkiRotaunit RU-200 (40 kV, 150mA) were used. The diffraction pattern was obtained from the microdensitometer trace along the equatorial direction. NMR analyses of the fractionated samples The lH and were carried out on a JEOL GSX-270 spectrometer in the pulsed mode. The measurement conditionswere Fourier transform (FT) almost similar to those of the other report.12 The GPC chromatograms were recorded with an HLC-802A high-performance liquid chromatograph (Tosoh Co., Ltd.) at 38 "C equipped with a series of four columns of TSK gel and an RI-8 differential refractometer. The eluent was chloroform with a flow rate of 1 mL/min, and the polymer concentration was ca. 1% (wlv). The number-average molecular weight, was calibrated using polystyrenestandards and six different M,values of PHB samples (from 4.29 X 108 to 5.64 X 10)evaluated by GPC and a low-angle laser light scattering (GPC-LALLS)system.
Results and Discussion Figure 1shows typical WAXS photographs of P(3HB) and the copolymers P(3HB-co-45.7 mol % 3HV) and P(3HB-co-64.2mol % 3HV) (hereinafterthese copolymers are called samples I and 11, respectively) and P(3HB-co67.8 mol% 3HV) using a cylindrical camera. The latter copolymer shows only the P(3HV) phase, while samples I and I1 show the mixed pattern of P(3HB) and P(3HV) crystal phases. If both samples are mixtures of several copolymers having HV contents lower or higher than ca. 40 mol % ,13 it is natural that they show the mixed WAXS patterns of both P(3HB) and P(3HV) crystal phases. Therefore, we carried out the fractional precipitation methods for both samples I and I1 using a solution of an acetone-water system. The results of fractionation of both samples and characterization of the fractionated samples were listed in Table I. The composition of the HV component(mol % ) was estimated from lH NMR study. In Table I, we can see that both samples I and I1 are the mixtures of several copolymers having different compositions. In order to determine the randomness in sequence distribution, it is convenient to use a new parameter D defined as D = FwFBB/(F@Bv) (where the subscripts of B and V present the HB and HV units, respectively, and FXY presents the mole fraction of the XY sequence estimated from the 13CNMR spectrum), which has been proposed by Kamiya et al.12 If a copolymer is statistically random, D is close to 1and larger or smaller than 1for a block or an alternate copolymer,respectively. All samples fractionated with the concentration of aqueous acetone less than 95 76 are typical random copolymers because D values are close to 1. Figure 2 shows DSC heating curves for the fractionated samples. Sample 1-1shows a main melting peak at 174 OC,which corresponds to P(3HB) homopolymer, accompanied by several small peaks at lower temperatures. This implies that sample 1-1is a mixture of P(3HB) (major content) and a minor content of several copolymershaving different HV composition, which is the reason why sample 1-1 showed the very large D value and very low HV
0 1993 American Chemical Society
Macromolecules, Vol. 26, No.21, 1993
5810 Notes
I L
Figure 1. Typical WAXS photographs of P(3HB) (a), P(3HB-co-45.7 mol%, 3HV) (b), P(3HB-co-64.2mol % 3HV) (c), and P(3HBeo-67.8 mol % 3HV) id) using a cylindrical camera. Photographs of a and d show typical P(3HB) and P(3HV) crystal phases, respectively, while those of b and c show the mixed diffraction patterns of P(3HB) and P(3HV) crystals.
sample I 1-1 1-2 1-3 1-4 I1 11-1 11-2 11-3 11-4 11-5 11-6
Table I. Fractionation and Characterization of Samples 1 and I1 cnnc of aq fraction acetone (9%) (wt %) HV mol % T, ("0 M nx 106
MdM.
D
45.7 23.0 71.6 44.6 40.9 64.2 85.0 78.1 72.2 55.2 48.2 44.5
3.44 3.60 3.06 3.31 2.93 2.90 2.83 2.88 2.82 2.44 2.42 2.55
1.48 29.6 1.15 1.03 0.82
100
90 85 80 100 95 90 85 80 70
27 19 42 4 12 5 14 36 11 16
72
2.65 3.14 2.63 2.48 1.96 2.82 2.93 2.91 2.94 2.80 2.39 1.81
&(,132,114
82
70 69 94 95 93 85 79 73 71
1.61
2.26 1.30 0.93 1.06 1.09 0.91 11-5
I
1
- 1-1 1-2
I -L w I1 11- I 11-3
Figure 2. DSC heating curves of the fractionated samples I an 11. composition. Therefore, sample 1-1 should be excluded from the following discussion. All the other samples seem to be the copolymers of single composition, because they show single and relatively sharp melting peaks. Samples I-2,1-3, and 1-4 show peaks at 82,70,69 O C , respectively. The fractionated samples of the I1 series show single and sharp melting peaks at a temperature range from 95 t o 71 "C. It was observed that T, decreases to the minimum value around 69 O C and the content of HV mol % converges toacompositionofca.40% as theconcentrationofacetone decreases. The number-average molecular weight M,, of the fractionated sample slightly decreased with decreasing the concentration of acetone, while M n of the sample fractionated a t extremely low concentration of acetone, i.e., 80 or IO%, decreased considerably, reflecting the increment in solubility of the sample.
20
10
ze ~~
~.~~
30
7 8. -
Fimre mol % '. .~ ..~ 3. Tvoical WAXS nattems of P(3HB-co-34.2 ~~~
~
I ~~
.
~ ~
~~
~~
~~~~~~~~
3HVh P(3HR-co.67.8mol% 3HV)andthe fractionatedsamples 1-4,l-3.II.5,and 11-4. ThediffractionpeaksRI,B2,and Rt were assigned M the t020).(110),and (002)planesoftheP(3HB)lattice, and thepeaksV,,V2.andV,.rothe(L10).(020).and (2ll)planes ofthe P(3HV)lattice,respectively. All fourfractionatednamplen show the mixed patterns of P(3HB)and P(3HV)cNstal phases. Typical WAXS patterns of these samples are shown in Figure 3, where the samples of HV contents of 34.2 and 67.8 mol ob show P(3HB) and P(3HV) crystal phases, respectively. Sample I-lshowed theP(3HB)crystalphase, while samples 1-2,11-1, 11-2, and 11-3 showed the P(3HV)
Notes 6811
Macromolecules, Vol. 26, No. 21, 1993
crystal phase. All other samples 1-3, 1-4, 11-4, and 11-5 show the mixed pattern of both P(3HB) and P(3HV) crystalphases as shown in Figure 3 (sample11-6also showed the mixed pattern). The characteristic diffraction peaks of the P(3HV) crystal phase (denoted as peaks VI, VZ,and V3) increased in their intensities with increasing the HV content. In Figure 3, both samples1-4 and 11-4whose HV contents are lowest and highest show the smallest diffraction peaks of P(3HV) and P(3HB) crystal phases, respectively. Consequently, samples I and I1 were demonstrated to be mixtures of severalrandom copolymershaving different compositions. The fractionated samples whose HV content range was 40.9-55.2 mol % showed the coexistence of both P(3HB) and P(3HV) crystal phases. The crystal phase transition of is~dimorphism~ for P(3HB-co-3HV), therefore, occurred rather at the broad composition range from 40.9 to 55.2 mol % HV. We could ascertain this fact with some other fractionated copolymers of HV contents lying between this range, which always showed the same mixed patterns of P(3HB)andP(3HV)crystal phases (data not shown). At this region, the lattice indices of the P(3HB) phase expanded up to the maximum values for P(3HB-co-55.2mol % 3HV) ( a = 0.602 nm; b = 1.343nm; c = 0.604 nm) from the original indices of P(3HB) ( a = 0.576 nm; b = 1.320 nm; c = 0.596 nm),l4Jswhereas those of the P(3HV) phase remained almost unchanged because the side group of the HB unit is smaller than that of the HV unit,13 though Scandola et al. reported the P(3HV)
crystal lattice slightly contracts with an increasing amount of HV units!
References and Notes Holmes, P. A. Phys. Technol. 1985,16,32. Doi, Y.; Tamaki, A.; Kunioka, M.; Soga, K. Appl. Microbiol. Biotechnol. 1988,28,330. Bluhm, T. L.; Hamer, G. K.; Marcheeeault, R. H.; Fyfe, C. A.; Veregin, R. P. Macromolecules 1986,19, 2871. Kunioka, M.; Tamaki, A.; Doi, Y. Macromolecules 1989,22, 694. Bloembergen, S.; Holden, D. A.; Bluhm, T. L.; Hamer, G. K.; Marchessault, R. H. Macromolecules 1989,22, 1663. Scandola, M.; Ceccorulli, G.; Pizzoli, M.; Gazzano, M. Macromolecules 1992,25, 1405. Kamiya, N.; Sakurai, M.; Inoue, Y.; Chajb, R.; Doi, Y. Macromolecules 1991,24, 2178. Orb,W. J.; Bluhm, T. L.; Marchessault, R. H. Polym. Prepr. (Am. Chem. SOC.,Diu. Polym. Chem.) 1992,33,530. Allegra, G.; Marcheeeault, R. H.; Bloembergen, S. J. Polym. Sci., Polym. Phys. Ed. 1992, 30, 809. Kamiya,N.;Sakurai, M.; Inoue, Y.; ChqjS, R. Macromolecules 1991,24,3888. Repaske, R.; Rapaske, A. C. Appl. Enoiron. Microbiol. 1976, 32, 585. Kamiya, N.; Yamamoto, Y.; Inoue, Y.; ChOj8, R.; Doi, Y. M~C~c"k?CUk8 1989,22, 1676. Kunioka, M.; Tamaki, A.; Doi, Y. Macromolecules 1989,22, 694. Yokouchi,M.; Chatani, Y.; Tadokoro,H.; Teraniehi,K.;Tani, H. Polymer 1973,14,267. Comibert, J.; Marcheeeault, R. H.; Benoit, H.; Weill, G. Macromolecules 1970,3, 741.
